Determination of [D-Ala2,D-Leu5]enkephalin and the metabolites containing C-terminal D-leucine by high-performance liquid chromatography.

A high-performance liquid chromatographic procedure has been developed for the determination of [D-Ala2,D-Leu5]enkephalin (DADLE) and the fragments containing D-leucine in rat blood. The procedure was applied to the determination of blood levels of [3H-D-Leu5]DADLE and the C-terminal fragments after intravenous administration of [3H-D-Leu5]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO4 and acetonitrile.